All cells possess genetic programs that respond to various stresses, such as starvation, temperature, irradiation, and infection. Those programs that have evolved to repel cellular invaders recognize and respond to alien nucleic acids and other microbial products. One such program consists of innate immune defenses, which are activated by viral infections, leading to the synthesis of interferon (IFN). IFNs induce an antivirl state by inducing the expression of IFN-stimulated genes (ISGs). IFNs are vital to the antiviral response, yet we do not understand how the 1000-plus genes encoding ISG products establish an antiviral state. There is little known about the mechanisms of action of ISGs, and their target specificity. Experiments in this application utilize a well-characterized human pathogen, poliovirus, to identify inhibitory ISG products and study their mechanism of action. The ability of poliovirus to replicate in cultured cells treated with IFN is abolished by a single amino acid change in the viral 2Apro proteinase. Insertion of the 2Apro coding sequence into the genome of the IFN-sensitive encephalomyocarditis virus (EMCV) confers the ability to replicate in the presence of IFN. Our hypothesis is that the viral 2Apro protein antagonizes the antiviral activity of one or more ISG products. To address this hypothesis, we will first identify ISG products that inhibit poliovirus replication and their mechanism of action (aim 1). We will determine if the 2Apro protein antagonizes the activity of these ISG products, and identify the mechanism. Passage of recombinant EMCV expressing poliovirus 2Apro in high levels of IFN leads to selection of viral mutants with higher resistance to IFN. Mutations responsible for this phenotype will be identified, and the mechanisms by which they enhance replication in the presence of the cytokine will be determined. Non-neuronal cells of mice are protected from poliovirus infection by the ISG response. However, poliovirus replicates well in the brain and spinal cord, leading to muscle paralysis. A hypothesis to explain these observations is that neuronal tissues do not mount a protective ISG response, allowing poliovirus replication. Replication of the IFN-sensitive poliovirus 2Apro mutant Y88L in the human neuroblastoma cell line SK-N-SH is relatively insensitive to IFN, suggesting that these cells may be used as a model for understanding why the IFN response in the central nervous system (CNS) does not impair poliovirus replication. In aim 2 we will elucidate the differences in the antiviral state between HeLa and SK-N-SH cells. We will determine if the ISG products identified in aim 1 are induced in SK-N-SH cells in response to viral infection. If the ISG products are not induced we will determine if their overexpression protects the neuronal cells from viral infection. The results of the proposed experiments will identify inhibitory ISGs and their mechanisms of action. This information will provide new insights into the IFN-induced antiviral state, and may provide novel targets for enteroviral therapeutics.